miR-363-3p靶向<i>TWIST1</i>调控上皮间质转化抑制非小细胞肺癌A549细胞迁移和侵袭

doi:10.3877/cma.j.issn.2095-1221.2020.03.001

目的

探讨miR-363-3p靶向TWIST1调控非小细胞肺癌A549细胞迁移和侵袭的分子机制。

方法

将体外培养的A549细胞分为空白对照组（细胞未做任何处理）；模拟物对照组（转染miR-363-3p模拟物阴性对照）；模拟物组（转染miR-363-3p模拟物）；后期实验另设：模拟物+ pcDNA组（转染miR-363-3p模拟物和pcDNA3.1空载体质粒）；模拟物+ TWIST1组（转染miR-363-3p模拟物和pcDNA3.1-TWIST1过表达质粒）。采用RT-PCR检测细胞中miR-363-3p的表达，Western blot检测细胞中TWIST1蛋白和上皮间质转化（EMT）相关蛋白Vimentin、E-cadherin的表达，Transwell小室实验检测细胞的迁移和侵袭。双荧光素酶报告基因实验检测miR-363-3p和TWIST1的靶向关系。两组间比较采用独立样本t检验，多组间比较采用单因素方差分析，组间两两比较采用LSD-t检验。

结果

与空白对照组和模拟物对照组比较，模拟物组A549细胞中miR-363-3p表达水平（1.00±0.08、0.97±0.05比3.82±0.45）升高，TWIST1 mRNA （1.00±0.06、0.98±0.06比0.39±0.02）和蛋白的表达水平（0.81±0.05、0.78±0.06比0.42±0.02）降低，差异具有统计学意义（P均< 0.05）。与空白对照组和模拟物对照组比较，模拟物组A549细胞Vimentin蛋白表达水平（0.58±0.04、0.55±0.05比0.36±0.03）降低，E-cadherin蛋白的表达水平（0.22±0.02、0.25±0.03比0.47±0.03）增高，迁移细胞数（85.75±5.45、83.52±6.85比53.05±4.50）和侵袭细胞数（128.26±6.15、125.95±8.05比71.64±5.75）降低，差异有统计学意义（P均< 0.05）。与模拟物对照组比较，模拟物组细胞中Vimentin蛋白的表达水平（0.55±0.05比0.36±0.03）降低，而E-cadherin蛋白表达水平（0.25±0.03比0.47±0.03）升高，迁移细胞数（83.52±6.85比53.05±4.50）和侵袭细胞数（125.95±8.05比71.64±5.75）均减少（P均< 0.05）。TWIST1是miR-363-3p潜在的靶基因。与模拟物+ pcDNA3.1组比较，模拟物+ TWIST1组A549细胞中Vimentin蛋白的表达水平（0.32±0.02比0.42±0.03）升高，而E-cadherin蛋白表达水平（0.56±0.04比0.38±0.03）降低，A549细胞的迁移数（49.45±4.22比67.52±5.05）和侵袭数（72.45±5.73比108.35±6.56）增加（P均< 0.05）。

结论

miR-363-3p可通过靶向TWIST1调控EMT抑制A549细胞迁移和侵袭。

关键词: miR-363-3p, 非小细胞肺癌, EMT, 迁移, 侵袭, TWIST1

Objective

To explore the molecular mechanism of miR-363-3p targeting TWIST1 regulates migration and invasion of non-small cell lung cancer A549 cells.

Methods

A549 cells cultured in vitro were divided into the blank control group (without any treatment) , mimic control group (transfected with miR-363-3p mimic negative control) , mimic group (transfected with miR-363-3p mimic) . Different groups were set up in the later experiment, including mimic+pcDNA group (transfected with miR-363-3p mimic and pcDNA3.1 empty vector plasmid) , mimic+TWIST1 group (transfected with miR-363-3p mimic and pcDNA3.1 TWIST1 overexpression plasmid) .The expression of miR-363-3p was detected by RT-PCR. The expression of TWIST1 protein and epithelial-mesenchymal transition (EMT) -related proteins Vimentin and E-cadherin were detected by Western blot, and the migration and invasion of cells were detected by Transwell chamber. The targeting relationship between miR-363-3p and TWIST1 was examed by dual luciferase reporter gene assay. Independent sample t-test was used for comparison between the two groups, single factor analysis of variance was used for comparison between multiple groups, and LSD-t test was used for comparison between the two groups.

Results

Compared with the blank control group and mimic control group, the expression of miR-363-3p (1.00±0.08, 0.97±0.05 vs 3.82±0.45) in A549 cells of mimic group were increased, and the TWIST1 mRNA and protein expression levels were decreased (1.00 ±0.06, 0.98±0.06 vs 0.39±0.02) , (0.81±0.05, 0.78±0.06 vs 0.42±0.02) , the difference was statistically significant (P < 0.05) . Compared with the blank control group and the mimic control group, the expression level of Vimentin protein in the mimic group A549 cells as well as the number of migration cells (85.75±5.45, 83.52±6.85 vs 53.05±4.50) and invasive cells (128.26±6.15, 125.95±8.05 vs 71.64±5.75) were decreased (0.58±0.04, 0.55±0.05 vs 0.36±0.03) , while the expression level of E-cadherin protein was increased (0.22±0.02, 0.25±0.03 vs 0.47±0.03) , the difference was statistically significant (all P < 0.05) . Compared with the mimic control group, the expression level of Vimentin protein in the simulant group cells (0.55±0.05 vs 0.36±0.03) and the number of migrating cells (83.52±6.85 vs 53.05±4.50) as well as invasive cells (125.95±8.05 vs 71.64±5.75) were decreased, while the expression level of E-cadherin protein (0.25±0.03 vs 0.47±0.03) was increased (all P < 0.05) . TWIST1 is a potential target gene of miR-363-3p. Compared with the mimic+pcDNA3.1 group, the expression level of Vimentin protein in A549 cells in the mimic+TWIST1 group was increased (0.32±0.02 vs 0.42±0.03) , while the expression level of E-cadherin protein (0.56±0.04 vs 0.38±0.03) was decreased. The migration number (49.45±4.22 vs 67.52±5.05) and invasion number (72.45±5.73 vs 108.35±6.56) of A549 cells were increased (all P < 0.05) .

Conclusion

MiR-363-3p could inhibit migration and invasion of A549 cells by regulating EMT through targeting TWIST1.

Key words: MiR-363-3p, Non-small cell lung cancer, EMT, Migration, Invasion, TWIST1

表1 引物序列信息

基因	序列（5'→3'）	预期扩增产物长度（bp）
miR-363-3p	上游TGGGGTGAGAGGTGATAGAGG	64
?	下游CGGCGTCTGCTGAACAAATAC	?
U6（内参）	上游CTCGGTTCGGCAGCACA	94
?	下游AACGCTTCACGAATTTGCGT	?
TWIST1	上游CATGTCCGCGTCCCACTAG	224
?	下游TGTCCATTTTCTCCTTCTCTCG	?
β-actin（内参）	上游CAGAAGGAGATTACTGCTCTGGCT	170
?	下游TACTCCTGCTTGCTGATCCACATC	?

表1 引物序列信息

基因	序列（5'→3'）	预期扩增产物长度（bp）
miR-363-3p	上游TGGGGTGAGAGGTGATAGAGG	64
?	下游CGGCGTCTGCTGAACAAATAC	?
U6（内参）	上游CTCGGTTCGGCAGCACA	94
?	下游AACGCTTCACGAATTTGCGT	?
TWIST1	上游CATGTCCGCGTCCCACTAG	224
?	下游TGTCCATTTTCTCCTTCTCTCG	?
β-actin（内参）	上游CAGAAGGAGATTACTGCTCTGGCT	170
?	下游TACTCCTGCTTGCTGATCCACATC	?

图1 Western blot检测TWIST1蛋白的表达

表2 模拟物干预后A549细胞中miR-363-3p及TWIST1 mRNA和蛋白表达水平（±s，n = 3）

分组	miR-363-3p表达量	TWIST1 mRNA表达量	TWIST1蛋白表达量
空白对照组	1.00±0.08	1.00±0.06	0.81±0.05
模拟物对照组	0.97±0.05	0.98±0.06	0.78±0.06
模拟物组	3.82±0.45^ab	0.39±0.02^ab	0.42±0.02^ab
F值	114.067	142.223	65.215
P值	< 0.001	< 0.001	< 0.001

表2 模拟物干预后A549细胞中miR-363-3p及TWIST1 mRNA和蛋白表达水平（±s，n = 3）

分组	miR-363-3p表达量	TWIST1 mRNA表达量	TWIST1蛋白表达量
空白对照组	1.00±0.08	1.00±0.06	0.81±0.05
模拟物对照组	0.97±0.05	0.98±0.06	0.78±0.06
模拟物组	3.82±0.45^ab	0.39±0.02^ab	0.42±0.02^ab
F值	114.067	142.223	65.215
P值	< 0.001	< 0.001	< 0.001

图2 TWIST1 3' UTR与miR-363-3p的结合位点

图3 模拟物及抑制剂干预细胞后双荧光素酶相对活性

图4 Western blot检测Vimentin和E-cadherin蛋白的表达

表3 miR-363-3p过表达对A549细胞中Vimentin和E-cadherin蛋白的相对表达水平（±s，n = 3）

分组	Vimentin蛋白表达量	E-cadherin蛋白表达量
空白对照组	0.58±0.04	0.22±0.02
模拟物对照组	0.55±0.05	0.25±0.03
模拟物组	0.36±0.03^ab	0.47±0.03^ab
F值	25.620	76.227
P值	0.001	< 0.001

表3 miR-363-3p过表达对A549细胞中Vimentin和E-cadherin蛋白的相对表达水平（±s，n = 3）

分组	Vimentin蛋白表达量	E-cadherin蛋白表达量
空白对照组	0.58±0.04	0.22±0.02
模拟物对照组	0.55±0.05	0.25±0.03
模拟物组	0.36±0.03^ab	0.47±0.03^ab
F值	25.620	76.227
P值	0.001	< 0.001

图5 倒置显微镜下观察miR-363-3p过表达对A549细胞迁移和侵袭的影响（结晶紫染色，×200）

表4 miR-363-3p过表达对A549细胞侵袭和迁移的影响（±s，n = 3）

分组	迁移细胞数（个）	侵袭细胞数（个）
空白对照组	85.75±5.45	128.26±6.15
模拟物对照组	83.52±6.85	125.95±8.05
模拟物组	53.05±4.50^ab	71.64±5.75^ab
F值	30.990	68.106
P值	< 0.001	< 0.001

表4 miR-363-3p过表达对A549细胞侵袭和迁移的影响（±s，n = 3）

分组	迁移细胞数（个）	侵袭细胞数（个）
空白对照组	85.75±5.45	128.26±6.15
模拟物对照组	83.52±6.85	125.95±8.05
模拟物组	53.05±4.50^ab	71.64±5.75^ab
F值	30.990	68.106
P值	< 0.001	< 0.001

图6 倒置显微镜下观察TWIST1对miR-363-3p过表达的A549细胞EMT、迁移和侵袭的影响（结晶紫染色，×200）

图7 Western blot检测Vimentin和E-cadherin蛋白表达

表5 TWIST1对miR-363-3p过表达的A549细胞EMT、迁移和侵袭的影响（±s，n = 3）

分组	Vimentin蛋白表达量	E-cadherin蛋白表达量	迁移细胞数（个）	侵袭细胞数（个）
模拟物+pcDNA3.1组	0.32±0.02	0.56±0.04	49.45±4.22	72.45±5.73
模拟物+TWIST1组	0.42±0.03	0.38±0.03	67.52±5.05	108.35±6.56
t值	4.804	6.235	4.756	7.139
P值	0.009	0.003	0.009	0.002

表5 TWIST1对miR-363-3p过表达的A549细胞EMT、迁移和侵袭的影响（±s，n = 3）

分组	Vimentin蛋白表达量	E-cadherin蛋白表达量	迁移细胞数（个）	侵袭细胞数（个）
模拟物+pcDNA3.1组	0.32±0.02	0.56±0.04	49.45±4.22	72.45±5.73
模拟物+TWIST1组	0.42±0.03	0.38±0.03	67.52±5.05	108.35±6.56
t值	4.804	6.235	4.756	7.139
P值	0.009	0.003	0.009	0.002

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Effects of MiR-363-3p targeting TWIST1 on the migration and invasion of A549 cells by regulating epithelial-mesenchymal transition

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